The goal of our research program is to develop therapies for inherited retinal diseases that support long-term photoreceptor function and survival. Three approaches have dominated efforts to develop effective for these diseases: corrective gene, anti-apoptotic, and neurotrophic therapies. The benefits obtained from these therapies have been relatively short-lived, many slowing but not preventing degeneration of the photoreceptor cells. The research outlined in this proposal is designed to determine if the therapeutic benefits of highly targeted combination treatments pairing corrective gene therapy with either anti-apoptotic or neurotrophic therapy are significantly greater than those obtained using corrective gene therapy alone. We will conduct these experiments in the cone-rich retinas of the GUCY1*B model of LCA1, a model system that is not only an excellent model for LCA1 but also provides a unique opportunity to examine the effects of combination treatments on cone cells. This project is divided into two research aims: AIM 1 - Develop dual-promoter, self inactivating, insulated lentiviral vectors capable of delivering multiple therapeutic transgenes to specific retinal cell types;AIM 2 -To test the hypothesis that degeneration of photoreceptors that have been treated with targeted corrective gene therapy can be prevented by combining corrective gene therapy with either targeted anti-apoptotic or targeted neurotrophic therapies. For Aim 1, dual-promoter vectors will be constructed that restrict expression of their therapeutic cargos to (1) cone and rod cells, (2) rod cells alone, and (3) M[unreadable]ller cells using cell-specific promoters. Vectors with the desired cellular expression characteristics will be modified to deliver corrective (RetGC1), anti-apoptotic (X-linked inhibitor of apoptosis, XIAP), and neurotrophic (rod derived cone viability factor, RdCVF;glial-derived neurotrophic factor, GDNF) therapies. Each of the therapeutic proteins, or the vectors carrying them, will be tagged with a unique fluorescent protein that we will use to analyze the effects of these treatments on photoreceptor cell survival. The vectors will be packaged into lentivirus and their expression characteristics will be analyzed in vivo using fluorescent microscopy, immuno-histochemistry, RT-PCR and western blot. For Aim 2, combination treatments pairing RetGC1 with either XIAP, RdCVF, or GDNF will be delivered to the retinas of GUCY1*B animals and the ability of these treatments to restore function to and promoter survival of photoreceptors will be evaluated using visual behavior assays, electroretinography, fluorescent microscopic analyses of retinal whole mounts, and molecular and protein analyses techniques. The fluorescent photomontages of whole mounts of the treated retinas will be analyzed using standard cell count methods and nearest neighbor and spatial clustering analyses techniques. The results of all of these analyses will permit us to determine if the combination treatments are effective in increasing survival of photoreceptor cells and if there is evidence of synergy between the delivered therapies that enhances photoreceptor survival. PUBLIC HEALTH RELEVANCE: Inherited retinal diseases that affect cone cells are among the most debilitating human retinal diseases. The research described in this proposal is designed to examine the ability of new combination treatments consisting of corrective gene, anti-apoptotic, and neurotrophic therapies to support and prevent degeneration of cone and rod cells affected by inherited retinal disease. This research will be carried out using an animal model of inherited retinal disease that possesses a cone-rich retina.